The methylene blue staining procedure is used to measure yeast viability based on the assumption that the methylene blue will enter the cells and be broken down by living yeast cells that produce the enzymes which breaks down methylene blue, leaving the cells colourless. The non- viable cells do not produce this enzyme (or enzymes) and as such the methylene blue that enters the cells are undegraded causing the cells to remain coloured (the oxidized form concentrates intracellularly).
The coloured and colourless cells are then counted using a haemocytometer and the number of viable and nonviable cells determined in a given area, the result would be then used to estimate the number of cells in the original sample. This is an easy, quick and cheap method to determine the amount of viable yeast present in a sample though this is not the best method for a number of reasons. A major reason is that methylene blue rapidly becomes toxic to the yeast and as such preparations should be examined within 10 minutes of preparation.
The older the cells become, the less likely that they are to take up the methylene blue dye from solution since as the yeast age, they deposit lipid and/or sugar in their cell membrane (in the form of free sterols[predominantly ergosterol and zymosterol with minor portions of lanosterol and fecosterol] and phospholipids[phosphotidylcholine and phosphotidylethanolamine with minor portions of phosphotidylinositol, phosphotidylserine and phosphotidyl-glycerol]) as a survival mechanism to protect their internal mechanisms from the buildup of waste in the external environment.
This means that cells which are not viable would not take up the dye and due to their age and not their ability to break down the methylene blue (as a result of their viability) would remain colourless and be determined to be viable (a false positive). The test itself is also not very accurate since yeast might not be evenly distributed in the original sample and depending on the sample taken for determination , may yield higher or lower viability counts than are really present in the original sample.
This viability count and cell count is absolutely essential since the yeasts are the producers of the ethanol through the breakdown of sugars (catabolism of sugars by glycolysis to pyruvate, which is then converted to CO2 and ethanol) present in the wort/must (the pitching rate is a measure of the number of viable yeast present in a particular stock being used for fermentation). These processes can only be performed by viable yeast cells and consequently the greater the umbers of viable yeasts in the sample, the higher the rate of ethanol production. The expected yield of ethanol (alcohol) can also be calculated based on the number of viable yeast present in a particular stock being used for fermentation (along with the sugar present in the wort). This is very important in industrial alcohol production to determine the required alcohol content of the final product and the viability of the yeast pitched must be greater then 85% or it is not used.
